The genome sequence of a muscid fly, Hydrotaea cyrtoneurina (Zetterstedt, 1845)

We present a genome assembly from an individual female Hydrotaea cyrtoneurina (muscid fly; Arthropoda; Insecta; Diptera; Muscidae). The genome sequence is 575.2 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 19.6 kilobases in length.


Background
Hydrotaea cyrtoneurina (Zetterstedt, 1845) is a representative of the muscid genus Hydrotaea Robineau-Desvoidy, 1830 (Diptera: Muscidae).The majority of males of Hydrotaea are known from a peculiar foreleg modification, potentially used for grasping females during mating (Gregor et al., 2002).Larvae are facultative carnivores feeding on decomposing organic tissues or preying on concomitant species.However, some species are obligatory predators with reduced number of free-living larval instars (Skidmore, 1985).
Hydrotaea cyrtoneurina distribution is restricted to the Palaearctic region, where it is widespread throughout Europe and eastwards to Siberia, India and the Korean Peninsula (Pont, 1986;Sorokina & Pont, 2010).It is a frequently occurring insect throughout the Archipelago of Britain and Ireland, with flight period mostly from April to October (d'Assis Fonseca, 1968).Adults are closely associated with badger setts, have been reported from fox faeces (Gregor et al., 2002), and are attracted to carrion and decomposing animal tissues (Grzywacz et al., 2017).During carrion succession experiments, it has been recognised significantly associated with bloated stage of decomposition (Matuszewski et al., 2010).The species lacks breeding records from animal carrion and human cadavers (Grzywacz et al., 2017;Matuszewski et al., 2008), yet larvae, potentially facultative predators with three fee-living larval stages, successfully develop on decomposing animal tissues (Grzywacz, 2013).
The genome presented here will serve as a source of information for phylogenomic purposes and may be used in comparative genomic studies to answer questions regarding biological adaptations in Hydrotaea species.
We present a chromosomally complete genome sequence for Hydrotaea cyrtoneurina, based on one female specimen from Wytham Woods, as part of the Darwin Tree of Life Project.This project is a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.

Genome sequence report
The genome was sequenced from one female Hydrotaea cyrtoneurina (Figure 1) collected from Wytham Woods, Oxfordshire, UK (51.77,.A total of 30-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 37 missing joins or mis-joins and removed 3 haplotypic duplications, reducing the scaffold number by 45.24%. The final assembly has a total length of 575.2 Mb in 22 sequence scaffolds with a scaffold N50 of 93.0 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.93%) of the assembly sequence was assigned to 6 chromosomal-level scaffolds, representing 6 autosomes.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition and nucleic acid extraction
A female Hydrotaea cyrtoneurina (specimen ID Ox001224, ToLID idHydCyrt1) was collected from Wytham Woods, Oxfordshire (biological vice-county Berkshire), UK (latitude 51.77, longitude -1.31) on 2021-04-19 using a net.The specimen was collected and identified by Steven Falk (independent researcher) and preserved on dry ice.In sample preparation, the idHydCyrt1 sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue from the thorax was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a).HMW DNA was extracted using the Automated MagAttract v1 protocol (Sheerin et al., 2023).DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30 (Todorovic et al., 2023).Sheared DNA was purified by solidphase reversible immobilisation (Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.
Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from abdomen tissue of idHydCyrt1 in the Tree of Life Laboratory at the WSI using the RNA Extraction: Automated MagMax™ mirVana protocol (do Amaral et al., 2023).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from head tissue of idHydCyrt1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016)   as described previously (Howe et al., 2021).Manual curation was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Software tool Version
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.
The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Andrés López Rubio
Tecnológico de Antioquia -University Institute, Antioquia, Colombia This article presents a genome sequence for Hydrotaea cyrtoneurina, a muscid fly with paleartic distribution.All the materials and method using the Pacific Biosciences platform were clear, as well as the assembly stratergies.
For future studies, have you considered the enrichment of this genome by including specimens at different locations along the species distribution.?
Is the rationale for creating the dataset(s) clearly described?

William Reid
Biochemistry, SUNY at Buffalo, Buffalo, New York, USA In the data note "The genome sequence of a muscid fly, Hydrotaea cyrtoneurina (Zetterstedt, 1845)", Falk et al present the genome assembly for Hydrotaea cyrtoneurina using PacBio long read sequencing and HiC scaffolding to reconstruct six autosomes.For their strategy, they divided the animal into the three soma, and used the head, thorax, and abdomen for the starting material for the HiC, genomic, and transcriptomic libraries, respectively.The abdomen would contain much of the metabolically active fat body (as well as other tissues) and be a good choice for the RNA library.Further, the omission of the abdomen from the genomic library avoids problems with the mating status of the female for the genomic assembly.Overall, they followed the standard pipeline for the Tree of Life consortium, which also has all of the protocols submitted to protocols.io.All data links are valid and available.
Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Insect transgenics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of Hydrotaea cyrtoneurina, idHydCyrt1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 575,214,542 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (150,892,273 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (92,963,629 and 81,795,257 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the diptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idHydCyrt1_1/dataset/idHydCyrt1_1/snail.

Figure 3 .
Figure 3. Genome assembly of Hydrotaea cyrtoneurina, idHydCyrt1.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idHydCyrt1_1/dataset/idHydCyrt1_1/blob.

Figure 4 .
Figure 4. Genome assembly of Hydrotaea cyrtoneurina, idHydCyrt1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idHydCyrt1_1/dataset/idHydCyrt1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Hydrotaea cyrtoneurina, idHydCyrt1.1:Hi-C contact map of the idHydCyrt1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=C1KA3sKITRG3i66TjhBUaQ.

Darwin Tree of Life Project Sampling Code of Practice', which
can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in

Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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